The present invention relates to a process for microbial cleavage of organic Cxe2x80x94N bonds and biodenitrogenation of nitrogen-containing organic carbonaceous material thereby. More specifically, total nitrogen from a hydrocarbon-containing medium may be reduced, as well nitrogen may be removed from the hydrocarbon molecule or from a fossil fuel. Microorganisms which have the ability of selective cleavage of organic Cxe2x80x94N bonds, particularly microorganisms such as Pseudomonas ayucida strain No PTA-806 can be used in the process that is the subject of this invention. The process of the invention is particularly useful in removal of organic nitrogen from fossil fuels, such as nitrogen-containing coal, petroleum oil, lignite and derived synthetic fuels while retaining the calorific value of the fuel.
The quality of petroleum is progressively deteriorating as the highest quality petroleum deposits are preferentially produced. Consequently, the concern about the concentration of compounds/contaminants such as sulfur, nitrogen, and metals in petroleum will intensify. These contaminants are not only contributors to environmental pollution resulting from the combustion of petroleum, but also interfere with the processing of petroleum by poisoning catalysts and contributing to corrosion. The selective removal of contaminants from petroleum while retaining the fuel value is a difficult technical challenge.
The selective removal of sulfur from dibenzothiophene with the aid of a bacterium useful for cleaving Cxe2x80x94S bonds is taught in U.S. Pat. No. 5,002,888.
U.S. Pat. No. 5,297,625 teaches a method for preparing, isolating and utilizing a microorganism which can metabolize crude oils and other high molecular weight hydrocarbons as a source of energy, and emulsify heavy crudes under the extreme conditions existing in oil reservoirs.
Biorefining of petroleum is therefore a technology soon being commercialized and which may be very promising.
The removal of nitrogen and metals from petroleum is a further potential use of biocatalysts, but so far this area of research has received very little attention.
Quinoline is perhaps the most widely studied organonitrogen compound as regards biodegradation, and quinoline is considered to be representative of many organonitrogen compounds typically found in petroleum. Many aerobic and anaerobic microbial cultures have been found that can degrade quinoline. The majority, if not the entirety, of microbial cultures described in the literature that metabolize quinoline do so by fully degrading it, and can therefore utilize quinoline as a sole source of carbon, energy, and nitrogen.
Shukla, Onkar P., in xe2x80x9cMicrobial Transformation of Quinoline by a Pseudomonas sp.xe2x80x9d, Applied and Environmental Microbiology, vol 51, June 1986, p. 1332-1342, reports that a Pseudomonas sp isolated from sewage by enrichment culture on quinoline metabolized this substrate by a novel pathway involving 8-hydroxycoumarin. Such microorganism utilizes quinoline as the sole source of carbon, nitrogen, and energy.
Schwarz, G. et al, in xe2x80x9cMicrobial Metabolism of Quinoline and Related Compounds. I. Isolation and Characterization of Quinoline-Degrading Bacteriaxe2x80x9d, System. Appl. Microbiol. 10, 185-190 (1988) report that from soil, water and activated sludge 16 bacterial strains were isolated which are able to use quinoline as sole source of carbon and nitrogen. Of the 16 bacterial strains investigated, 13 could be allocated to the genus Pseudomonas. These bacteria are Gram-negative, straight to slightly curved, motile rods, which on HNB-agar form yellowish to cream-colored, circular, smooth or partially rough colonies. The species identified are Pseudomonas putida Biovars A and B, Pseudomonas fluorescens and Pseudomonas testosteroni. It is reported that growth on 2-hydroxyquinoline is common to all strains, which were investigated. With several Pseudomonas species the degradation of quinoline has been studied and 2-hydroxyquinoline was found to be the first intermediate in the degradation pathway.
Grant, D. J. W. et al. In xe2x80x9cDegradation of quinoline by a soil bacteriumxe2x80x9d, Microbios 1976, 15, p. 177-189, report that from garden soil a bacterium was isolated which grew aerobically in mineral salts medium with quinoline as sole C source and NH4+ as N source. During growth with quinoline, 2-hydroxyquinoline accumulated in the culture fluid and later disappeared. 2,6-Dihydroxyquinoline is probably the next intermediate in quinoline biodegradation by aerobic organisms examined in this report since whole cells oxidize it rapidly and completely. Aromatic ring cleavage under aerobic conditions almost invariably follows the formation of a compound with two hydroxyl groups attached to a ring in positions o- or p- to each other.
O""Loughlin, E. J. et al. In xe2x80x9cIsolation, Characterization and Substrate Utilization of a Quinoline-Degrading Bacteriumxe2x80x9d, International Biodeterioration and Biodegradation (1996), 107-118 report a Gram (+) rod-shaped organism identified as a Rhodococcus sp. capable of growth utilizing quinoline as the dominant carbon, energy, and nitrogen source. The isolate, designated as Rhodococcus sp. Q1 was also capable of growth on 2-hydroxyquinoline, pyridine, 2,3-dimethyl pyridine, catechol, benzoate, and protocatechuic acid, suggesting a diverse capacity for aromatic ring degradation. Although ring nitrogen was released into the growth medium as ammonium, quinoline degradation was not limited by the availability of inorganic N. A degradation product identified as 2-hydroxyquinoline was identified on the basis of several spectroscopic analyses.
Thus, although the cited literature mentions microbial cultures able to metabolize quinoline by fully degrading it, the use of such cultures in a petroleum biorefining application would require that nitrogen be selectively removed from quinoline leaving the carbon and the calorific value of the molecule intact.
As related in the literature, the metabolic pathways utilized by various aerobic quinoline-degrading microorganisms were shown to initiate the degradation of quinoline by selectively oxidizing and removing nitrogen from quinoline.
While the biodegradation of quinoline has been reasonably well studied there is very little information concerning the use of quinoline-degrading microorganisms to remove nitrogen from petroleum. On the other hand, several quinoline-degrading Pseudomonas were found to have no ability to remove significant levels of nitrogen from crude oil or asphaltene fractions of petroleum (Aislabie et al. 1990, xe2x80x9cMicrobial Degradation of Quinoline and Methylquinolinexe2x80x9d, Appl. Environ. Microbiol. 56:345-351).
Therefore there is the need to isolate aerobic microbial cultures capable of utilizing quinoline as a nitrogen source, but incapable of utilizing quinoline as a carbon source, the ability of such cultures to selectively remove nitrogen from petroleum, as well as a process for microbial cleavage selectively of organic Cxe2x80x94N bonds and reduction of nitrogen from nitrogen-containing organic carbonaceous material thereby, such goal being achieved by the present invention.
It is an object of the present invention to provide a microbial process for selective removal of organically bound nitrogen from nitrogen-containing organic carbonaceous materials.
It is another object of the present invention to provide a microbial process for selective nitrogen removal from organic nitrogen-containing fossil and fossil derived fuels.
It is yet another object of the present invention to provide a microorganism and process capable of specific cleavage or formation of Cxe2x80x94N bonds in reactions of organic carbonaceous materials, such as in organic synthesis.
It is still another object of the present invention to provide a microorganism and process for specific nitrogen removal from quinoline resulting in the primary organic product of 8-hydroxycoumarin.
It is still another object of the present invention to provide a process using a microorganism which is stable and retains its ability to selectively cleave Cxe2x80x94N bonds under conditions suitable for an industrial process for cleavage of organic Cxe2x80x94N bonding.
The above and other subjects and advantages, as will become apparent upon reading this description, can be achieved by contacting a nitrogen-containing organic carbonaceous material with a microorganism which has the ability of selective cleavage of organic Cxe2x80x94N bonds and production of 8-hydroxycoumarin when grown in a growth medium comprising mineral nutrients and an assimilable source of carbon in the presence of oxygen and the substantial absence of a nitrogen-containing compound except the nitrogen-containing organic carbonaceous material. Pseudomonas ayucida strain No PTA-806 having the above properties is particularly preferred.
The above and other objects and advantages, as will become apparent upon reading this description, have been achieved by the pure culture of a microorganism which has been isolated and subjected to processes as set forth in further detail and identified as Pseudomonas ayucida. The culture has been deposited under the Budapest Treaty with American Type Culture Collection, 10801 University Blvd, Manassas, Va. 20210-2209, USA, and assigned No PTA-806.
Pseudomonas ayucida No PTA-806 may be prepared by inoculating environmental samples derived from sites having present materials of Cxe2x80x94N bonding desired to be cleaved, a growth medium comprising mineral nutrients, an assimilable source of carbon, and in substantial absence of a nitrogen-containing compound, except compounds having nitrogen present only in Cxe2x80x94N bonding of the type desired to be cleaved; growing the bacterial culture in the presence of oxygen at temperatures about 25xc2x0 C. to about 35xc2x0 C. and in the substantial absence of a nitrogen-containing compound except compounds having nitrogen present only in Cxe2x80x94N bonding of the type desired to be cleaved for sufficient time to selectively produce Pseudomonas ayucida No PTA-806 which has the property of nitrogen metabolism by selective cleavage of Cxe2x80x94N bonds in organic carbonaceous materials.
Nitrogen content of nitrogen-containing organic carbonaceous material may be reduced by contacting such nitrogen-containing organic carbonaceous material with the microorganism Pseudomonas ayucida strain No PTA-806. The process is especially suitable for use where the nitrogen-containing material is a coal, shale oil or hydrocarbon oil.
Continuous growth of Pseudomonas ayucida strain No PTA-806 in the presence of nitrogen-containing shale oil results in the removal of more than 50 wt percent, and preferably more than 68 wt percent, of the organically bound nitrogen as quinoline.
The process for reducing the nitrogen content of the nitrogen-containing organic carbonaceous material occurs by cleavage of organic Cxe2x80x94N bonds by the microorganism Pseudomonas ayucida strain No PTA-806. The organic nitrogen selective microorganism Pseudomonas ayucida No PTA-806 has the ability to selectively reduce the nitrogen content of nitrogen-containing organic carbonaceous material by cleavage of organic Cxe2x80x94N bonds by production of 8-hydroxycoumarin when grown in a growth medium comprising mineral nutrients and an assimilable source of carbon in the substantial absence of a nitrogen-containing compound except the nitrogen-containing organic carbonaceous material, and in the presence of oxygen at temperatures about 25xc2x0 C. to about 35xc2x0 C. Derivative microorganisms of Pseudomonas ayucida No PTA-806 that retain the ability to selectively cleave Cxe2x80x94N bonds can also be used in the same fashion for the reduction of the nitrogen content of carbonaceous material.
The above and other objects and advantages have also been achieved by the microorganism which has been isolated and subjected to processes as set forth in further detail and identified as Pseudomonas ayucida. The culture has been deposited under the Budapest Treaty with American Type Culture Collection, 10801 University Blvd, Manassas, Va. 20110-2209, USA, and assigned No PTA-806. This microorganism, its production and use for selective cleavage of organic Cxe2x80x94N bonds is more fully described in a copending, commonly owned US patent application, which is incorporated herein by reference in its entirety.